It is being increasingly recognized that multiple pathways contribute to necrotic cell death in a highly regulated fashion and are amenable to specific interventions. Processes that have been implicated include necrotic cell death related to development of the mitochondrial permeability transition (MPT) regulated by cyclophilin D (CypD), necroptosis mediated by receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and sensitive to inhibition by necrostatin-1, and pyroptosis resulting from activation of caspases 1 and 11. The ultimate downstream event required for several of the necrotic, immunogenic processes is glycine-sensitive opening of a plasma membrane channel. Preliminary studies for this proposal show that an additional pathway of iron-mediated cell death termed 'ferroptosis' may account for the iron-related cell injury involved in several common and clinically relevant forms of acute kidney injury (AKI). Ferroptosis is subject to modulation by novel small molecule inhibitors that emerged from chemical library screening, ferrostatins, as well as by other lipophilic antioxidants and by inhibition of NADPH oxidase. The objectives of this proposal are to further investigate and clarify the mechanisms of ferroptosis and its expression in freshly isolated kidney proximal tubules ex vivo, its impact on AKI in vivo, and its interactions with necroptosis and cyclophilin D pathways of regulated necrosis to address the hypothesis that regulated necrosis resulting from additive effects of ferroptosis, induction of the MPT mediated by CypD, and necroptosis contributes to tubule cell killing during AKI and is a target for pharmacological intervention. Studies will utilie tubules from rabbits and mice subjected to injury ex vivo and in vivo models of mouse AKI along with pharmacologic modulators and mutant mice deficient in CypD or RIPK3, newly developed mice deficient in both CypD+RIPK3 or caspase+RIPK3, and mice deficient in NADPH oxidase 4. Tubules isolated by collagenase digestion will be subjected to oxidant and iron-induced injury by tert-butylhydroperoxide or by hydroxyquinoline plus ferrous ammonium sulfate, or to hypoxia/reoxygenation. Lethal membrane damage will be quantitated as the final endpoint, and, at time points preceding that damage, measurements of ATP, mitochondrial membrane potential, reactive oxygen species production, and lipid peroxidation will be made to assess the mechanisms of this injury and its modification by ferrostatins, pharmacologic inhibitors of the MPT and necroptosis, and absence of pathway proteins. AKI will be produced in vivo by glycerol-induced rhabdomyolysis, clamp ischemia, or cisplatin followed by assessment of renal function and structural changes. These studies will further elucidate the role of newly characterized forms of regulated necrosis during AKI and provide insight into approaches for ameliorating them and the organ failure that results.